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  7. Concepts of Protein Technology and applications (UA_2050FBDBMW_2324)

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Summary Concepts of Protein Technology and Applications (14/20)
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  • Vak
  • Concepts of Protein Technology and applications (UA_2050FBDBMW_2324)
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  • Universiteit Antwerpen (UA)

This document serves as a comprehensive summary combining the key insights from the presentations of Professors Xaveer van Ostade and Kurt Boonen. The summary consists of their slides, enriched with my personal notes.

Laatste update van het document: 11 maanden geleden

Voorbeeld 4 van de 155  pagina's

Document informatie

  • 18 december 2023
  • 28 december 2023
  • 155
  • 2023/2024
  • Samenvatting

Onderwerpen

  • concepts
  • proteomics
  • mass spectrometry
  • 1st master
  • xaveer van ostade
  • kurt boonen
  • ms

Geschreven voor

  • Universiteit Antwerpen (UA)
  • Biomedische Wetenschappen
  • Concepts of Protein Technology and applications (UA_2050FBDBMW_2324)
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Voorbeeld van de inhoud

CONCEPTS OF PROTEIN TECHNOLOGY
AND APPLICATIONS
INHOUDSOPGAVE

1.1 Definition proteomics .......................................................................................................................... 1

1.2 Why proteomics? ................................................................................................................................ 1
1.2.1 Compared to genomics, proteomics is “the real thing” ....................................................................... 1
1.2.2 mRNA vs protein profiling ................................................................................................................... 2
1.2.3 More (6-8) proteins/gene .................................................................................................................... 2
1.2.3.1 Posttranslational modification ........................................................................................................ 2
1.2.3.2 Alternative splicing à isoforms ...................................................................................................... 3
1.2.4 Protein interaction networks ............................................................................................................... 3
1.2.5 Cellular localization ............................................................................................................................. 3

1.3 Proteomics as part of systemsbiology .................................................................................................. 4

1.4 The different faces of proteomics ........................................................................................................ 4

1.5 Identification of proteins: principles .................................................................................................... 5
1.5.1 Sample preparation ............................................................................................................................. 6

1.5.2 Protein peptide separation .................................................................................................................. 6
1.5.2.1 Increasing separation capacity (peak capacity) ............................................................................... 7
1.5.2.2 2D-Electrophoresis (2D-PAGE) ........................................................................................................ 7
1.5.2.3 Chromatography ............................................................................................................................. 8
1.5.3 Analysis: mass spectrometry ............................................................................................................... 9
1.5.4 Protein identification by data analysis .............................................................................................. 10

1.6 Workflows for ‘shotgun’ proteomics .................................................................................................. 10

2.1 Proteomics introduction .................................................................................................................... 11

2.1.1 Part of life sciences ............................................................................................................................ 11
2.1.2 from genome to proteome ................................................................................................................ 12
2.1.2.1 Why proteomics? .......................................................................................................................... 12
2.1.2.2 The proteome is very complex! ..................................................................................................... 12
2.1.2.3 From genome to proteome ........................................................................................................... 13
2.1.3 Protein variants in disease................................................................................................................. 13

2.2 Overview: what do we do with a complex sample? ........................................................................... 14


1

,2.3 Sample preparation steps .................................................................................................................. 15

2.3.1 Defining your research question ........................................................................................................ 16
2.3.2 Literature study ................................................................................................................................. 16

2.3.3 Sample collection............................................................................................................................... 17
2.3.3.1 Sample types ................................................................................................................................. 17
2.3.3.2 Sample degradation ...................................................................................................................... 17
2.3.4 Protein extraction – solubilization ..................................................................................................... 17
2.3.4.1 Protein extraction.......................................................................................................................... 17
2.3.4.2 Protein solubilization ..................................................................................................................... 20

2.3.5 Protein separation or purification ..................................................................................................... 22
2.3.5.1 Acetone precipitation .................................................................................................................... 22
2.3.5.2 Gelfiltration ................................................................................................................................... 22
2.3.5.3 Filtering.......................................................................................................................................... 22
2.3.5.4 SDS-PAGE....................................................................................................................................... 23
2.3.6 Reduction and alkylation ................................................................................................................... 23
2.3.7 Digestion............................................................................................................................................ 24
2.3.8 Peptides purification or selection ...................................................................................................... 25
2.3.9 Prefractionation ................................................................................................................................ 26

3.1 Introduction ...................................................................................................................................... 27

3.1.1 AA analyses and AA sequencing are two different techniques .......................................................... 27
3.1.2 What can Amino Acid Analysis (AAA) do for you? ............................................................................. 27

3.2 Sample Preparation for Amino Acid Analysis ..................................................................................... 28

3.3 AAA of proteins/peptides .................................................................................................................. 28

3.3.1 In general........................................................................................................................................... 28
3.3.2 Hydrolysis .......................................................................................................................................... 28

3.3.3 Separation of amino acids ................................................................................................................. 30
3.3.3.1 Paradigms ...................................................................................................................................... 30
3.3.3.2 Pre-column derivatization ............................................................................................................. 30
3.3.3.3 Post-column derivatization ........................................................................................................... 31
3.3.3.4 Pre- versus post-column derivatization ......................................................................................... 32

3.4 Amino acid sequencing ...................................................................................................................... 33
3.4.1 In general........................................................................................................................................... 33
3.4.2 Edman degradation ........................................................................................................................... 33
3.4.2.1 Insulin ............................................................................................................................................ 33
3.4.2.2 How does it work? ......................................................................................................................... 33
3.4.2.3 Cyclical estimation of yields .......................................................................................................... 34
3.4.2.4 Side products ................................................................................................................................. 34
3.4.2.5 Edman chemistry also disrupts AAs............................................................................................... 35
3.4.2.6 Positive aspects and drawbacks of Edman sequencing ................................................................. 35



2

, 3.4.3 Cleavage ............................................................................................................................................ 35
3.4.3.1 Chemical cleavage ......................................................................................................................... 35
3.4.3.2 Enzymatic cleavage ....................................................................................................................... 36

3.5 Carboxyterminal sequencing ............................................................................................................. 36
3.5.1 Carboxypeptidases ............................................................................................................................ 36
3.5.2 Chemical process ............................................................................................................................... 36

3.6 Single molecule sequencing ............................................................................................................... 37
3.6.1 Edman degradation ........................................................................................................................... 37
3.6.2 Microscale Edman degradation......................................................................................................... 37

4.1 Introduction ...................................................................................................................................... 39
4.1.1 Electrophoresis .................................................................................................................................. 39
4.1.2 2-dimensional gel electrophoresis ..................................................................................................... 39

4.2 Basic principles .................................................................................................................................. 39

4.2.1 In general........................................................................................................................................... 39
4.2.2 In practice .......................................................................................................................................... 40

4.3 First dimension: Isoelectric focusing (IEF)........................................................................................... 40
4.3.1 Isoelectric focusing ............................................................................................................................ 40
4.3.2 Protein charge ................................................................................................................................... 40
4.3.3 Creating a pH gradient for IEF ........................................................................................................... 41
4.3.3.1 Using carrier ampholytes .............................................................................................................. 42
4.3.3.2 IMMOBILIZED PH GRADIENTS ....................................................................................................... 42

4.3.4 2D-GE sample preparation ................................................................................................................ 43
4.3.5 Standard IP run for IEF and 2D-GE..................................................................................................... 44

4.4 Second Dimension: SDS-PAGE............................................................................................................ 44
4.4.1 SDS-PAGE ........................................................................................................................................... 44

4.4.2 Mass markers .................................................................................................................................... 45
4.4.3 PAGE matrix....................................................................................................................................... 46
4.4.3.1 Composition .................................................................................................................................. 46
4.4.3.2 Polymerization............................................................................................................................... 46
4.4.4 SDS-PAGE systems ............................................................................................................................. 46

4.5 Sample preparation ........................................................................................................................... 47
4.5.1 IEF preparation .................................................................................................................................. 47
4.5.2 Preparation of pH gradient gels ........................................................................................................ 47
4.5.3 IPG preparation for second dimension .............................................................................................. 47




3

, 4.6 Protein detection methods 2D-GE...................................................................................................... 48

4.6.1 Coomassie Brilliant Blue (CBB R-250) ................................................................................................ 48
4.6.2 Silver staining .................................................................................................................................... 49

4.6.3 SYPRO Ruby staining.......................................................................................................................... 49
4.6.4 2D-GE autoradiography..................................................................................................................... 49
4.6.5 Overview of staining options ............................................................................................................. 49

4.7 Analysis of 2D-gels ............................................................................................................................. 50
4.7.1 Image processing – digitalization ...................................................................................................... 50
4.7.2 Protein spot or ‘gradu’....................................................................................................................... 50

4.7.3 Complexities of 2D-GE ....................................................................................................................... 50

4.8 Differential in-gel electrophoresis (DIGE) ........................................................................................... 51
4.8.1 In general........................................................................................................................................... 51
4.8.2 Application ........................................................................................................................................ 51

4.8.3 Example ............................................................................................................................................. 52
4.8.4 DeCyder v. 6.5.................................................................................................................................... 52

4.9 2D-GE protein identification .............................................................................................................. 52

4.10 Take home points .............................................................................................................................. 52

5.1 Introduction ...................................................................................................................................... 53

5.2 Hydrophobic interactions .................................................................................................................. 53

5.3 Standard conditions for RP-HPLC ....................................................................................................... 54
5.3.1 Stationary phase................................................................................................................................ 54
5.3.2 Mobile phase ..................................................................................................................................... 55
5.3.2.1 Organic solvent .............................................................................................................................. 56
5.3.2.2 pH of mobile phase ....................................................................................................................... 58
5.3.2.3 Agents for ion-pairing .................................................................................................................... 58

5.4 Microcolumn Reverse Phase chromatography ................................................................................... 60
5.4.1 General .............................................................................................................................................. 60
5.4.2 Principle ............................................................................................................................................. 60

5.4.3 Adaptation of optimal flow rate ........................................................................................................ 61
5.4.4 Capacity and resolution of microcolumns ......................................................................................... 61
5.4.5 Adaptations of the HPLC system ....................................................................................................... 62

5.5 2D-LC ................................................................................................................................................. 63
5.5.1 Peak capacity and set-up of a 2D-LC ................................................................................................. 63



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